Staphylococcus aureus (S. aureus) is a major cause of human morbidity and mortality. Strains classified as pulsed-field gel electrophoresis type (PFGE) USA300 are particularly adept at causing skin and soft-tissue infections. A recent report identified methicillin-resistant S. aureus (MRSA) as the leading cause of infections presenting to emergency departments across the United States accounting for ~ 59% of skin and soft-tissue infections. Community-associated MRSA strain USA300 was identified as the causative agent in e 97% of these infections. The objective of this proposal is to pursue specific molecular mechanisms used by S. aureus at the host-pathogen interface. As community associated-MRSA strain USA300 is a major cause of skin infections, we examined the role of the SaeR/S two-component gene regulatory system in this strain using a murine model of skin and soft-tissue infection. A saeR/S isogenic deletion mutant of USA300 (USA300?saeR/S) demonstrated a significant decrease in incidence and severity of infection compared to USA300. In vitro oligonucleotide microarray and real-time reverse-transcriptase polymerase chain reaction assays identified numerous extracellular virulence genes down-regulated in USA300?saeR/S and direct binding of recombinant SaeR to a consensus sequence within virulence gene promoters. On the host side, we have determined that SaeR/S-influenced factors alter the local production of pro-inflammatory cytokines IFN? and TNF? during soft-tissue infections. Based on preliminary data, we hypothesize that virulence factors under the direct influence of SaeR/S modulate the severity of S. aureus skin infections. We will test this hypothesis with two specific aims. In Specific Aim 1 we will determine if SaeR directly regulates S. aureus virulence genes in vivo during skin infection. Specific roles of saeR/S-regulated virulence factors will be pursued by creating isogenic mutants and investigating their influence on staphylococcal pathogenesis. In Specific Aim 2, we will define the SaeR/S-specific host response by testing the hypothesis that SaeR/S-regulated factors decrease TNF? and increase IFN? to promote pathogenesis during staphylococcal skin infection. Collectively, this research will improve our understanding of the initial host-pathogen interactions by identifying specific S. aureus factors that promote skin infection and may potentially have implications for the use of immunomodulatory therapy to treat S. aureus infections. Given the diminishing effectiveness of our current antibiotics, it is clear we must improve understanding of molecular mechanisms fundamental to S. aureus virulence to control disease caused by this potentially life-threatening pathogen. PUBLIC HEALTH RELEVANCE: Staphylcoccus aureus (S. aureus) is a leading cause of morbidity and mortality worldwide. The emergence of hyper-infectious community-associated methicillin resistant (CA-MRSA) strains within the community is a major public health concern. Given the diminishing effectiveness of our current antibiotics, it is clear we must improve our understanding of molecular mechanisms fundamental to S. aureus pathogenesis in order to develop better methods to control/treat CA-MRSA disease and thus improve public health.